Journal: Acta Neuropathologica Communications

Article Title: The ESCRT-III protein VPS4, but not CHMP4B or CHMP2B, is pathologically increased in familial and sporadic ALS neuronal nuclei

doi: 10.1186/s40478-021-01228-0

Figure Lengend Snippet: VPS4, but not CHMP4B nor CHMP2B, is increased in C9orf72 and sALS postmortem motor cortex neuronal nuclei. a , b Maximum intensity projections from SIM imaging of CHMP4B, CHMP2B, and VPS4 in nuclei isolated from postmortem control, C9orf72 , and sALS motor ( a ) and occipital ( b ) cortex tissue. Genotype as indicated on left, brain region and antibody as indicated on top. c – e Quantification of CHMP4B ( c ), CHMP2B ( d ), and VPS4 ( e ) spots. n = 6 control, 6 C9orf72 , and 9 sALS cases, 50 NeuN + nuclei per line/brain region. One-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **** p < 0.0001. Scale bar = 5 μm

Article Snippet: Plasmids used are as follows: GFP (Addgene 54759), VPS4 GFP (Addgene 116924), and VPS4 E228Q GFP (Addgene 80351).

Techniques: Imaging, Isolation, Control, Comparison

Journal: Acta Neuropathologica Communications

Article Title: The ESCRT-III protein VPS4, but not CHMP4B or CHMP2B, is pathologically increased in familial and sporadic ALS neuronal nuclei

doi: 10.1186/s40478-021-01228-0

Figure Lengend Snippet: VPS4, but not CHMP4B nor CHMP2B, is increased in C9orf72 and sALS iPSN nuclei. a , b Maximum intensity projections from SIM imaging of CHMP4B, CHMP2B, and VPS4 in nuclei isolated from control, C9orf72 , and sALS iPSNs at day 18 ( a ) and 32 ( b ) of differentiation. Genotype as indicated on left, time point and antibody as indicated on top. c – e Quantification of CHMP4B ( c ), CHMP2B ( d ), and VPS4 ( e ) spots. n = 10 control, 10 C9orf72 , and 10 sALS iPSC lines, 50 NeuN + nuclei per line/time point. One-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. ** p < 0.01, **** p < 0.0001. Scale bar = 5 μm

Article Snippet: Plasmids used are as follows: GFP (Addgene 54759), VPS4 GFP (Addgene 116924), and VPS4 E228Q GFP (Addgene 80351).

Techniques: Imaging, Isolation, Control, Comparison

Journal: Acta Neuropathologica Communications

Article Title: The ESCRT-III protein VPS4, but not CHMP4B or CHMP2B, is pathologically increased in familial and sporadic ALS neuronal nuclei

doi: 10.1186/s40478-021-01228-0

Figure Lengend Snippet: Nuclear expression of VPS4 is dependent upon CHMP7 in C9orf72 and sALS iPSNs. a , c , e Maximum intensity projections from SIM imaging of CHMP4B ( a ), CHMP2B ( c ), and VPS4 ( e ) in nuclei isolated from control, C9orf72 , and sALS iPSNs following 2 week exposure to 5 μM scrambled control or CHMP7 ASO. Treatment as indicated on left, genotype and antibody as indicated on top. b , d , f Quantification of CHMP4B ( b ), CHMP2B ( d ), and VPS4 ( f ) spots. n = 5 control, 5 C9orf72 , and 5 sALS iPSC lines, 50 NeuN + nuclei per line/treatment. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. ** p < 0.01, **** p < 0.0001. Scale bar = 5 μm

Article Snippet: Plasmids used are as follows: GFP (Addgene 54759), VPS4 GFP (Addgene 116924), and VPS4 E228Q GFP (Addgene 80351).

Techniques: Expressing, Imaging, Isolation, Control, Comparison

Journal: Acta Neuropathologica Communications

Article Title: The ESCRT-III protein VPS4, but not CHMP4B or CHMP2B, is pathologically increased in familial and sporadic ALS neuronal nuclei

doi: 10.1186/s40478-021-01228-0

Figure Lengend Snippet: Overexpression of a dominant negative VPS4 increases nuclear POM121 spots but does not restore their distribution in C9orf72 and sALS iPSNs. a , c Maximum intensity projections from SIM imaging of VPS4 ( a ) and POM121 ( c ) in nuclei isolated from control, C9orf72 , and sALS iPSNs overexpressing GFP or GFP tagged VPS4 variants. Overexpression as indicated on left, genotype and antibody as indicated on top. Arrows ( c ) indicate uneven distribution of POM121 observed following overexpression of dominant negative VPS4 (VPS4 E228Q ) in ALS nuclei. b , d Quantification of VPS4 ( b ) and POM121 ( d ) spots. n = 4 control, 4 C9orf72 , and 4 sALS iPSC lines, 50 GFP + nuclei per line/overexpression. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. * p < 0.05, ** p < 0.01, **** p < 0.0001. Scale bar = 5 μm

Article Snippet: Plasmids used are as follows: GFP (Addgene 54759), VPS4 GFP (Addgene 116924), and VPS4 E228Q GFP (Addgene 80351).

Techniques: Over Expression, Dominant Negative Mutation, Imaging, Isolation, Control, Comparison

Journal: Pharmaceutics

Article Title: The PIK3CA H1047R Mutation Confers Resistance to BRAF and MEK Inhibitors in A375 Melanoma Cells through the Cross-Activation of MAPK and PI3K–Akt Pathways

doi: 10.3390/pharmaceutics14030590

Figure Lengend Snippet: Transduction efficiency of retroviral infection and cell growth analysis. ( A ) Western blot analysis of A375 H1047R compared with that of A375 PIK3CA and A375 pLNCX2 . ( B ) Protein levels of p-ERK, p-Akt, and total ERK; Akt was evaluated to verify the transduction efficiency. ( C ) Cell growth was evaluated by MTT assay at 12, 24, 48, and 72 h. All experiments were performed in triplicate. * p ≤ 0.05; ** p ≤ 0.01.

Article Snippet: The pLNCX2-PIK3CA wt and pLNCX2-PIK3CA H1047R were obtained by cloning PIK3CA wt and PIK3CA H1047R inserts from pBabe puro HA PIK3CA (Plasmid #12522—Addgene) and pBabe puro HA PIK3CA H1047R (Plasmid #12524—Addgene) plasmids, respectively, into pLNCX2 plasmid.

Techniques: Transduction, Infection, Western Blot, MTT Assay

Journal: Pharmaceutics

Article Title: The PIK3CA H1047R Mutation Confers Resistance to BRAF and MEK Inhibitors in A375 Melanoma Cells through the Cross-Activation of MAPK and PI3K–Akt Pathways

doi: 10.3390/pharmaceutics14030590

Figure Lengend Snippet: Dabrafenib and trametinib treatment of A375 pLNCX2 , A375 PIK3CA , and A375 H1047R cells. ( A ) Cell viability evaluated through the MTT assay after 72 h of dabrafenib treatment at different concentrations (0.01–0.1–1–10–100–1000 nM). ( B – D ) Western blot analyses of p-ERK, p-AKT, total ERK, and total AKT protein levels and their ratio in A375 transduced cells treated with dabrafenib (0.0–0.5–2 nM) for 48 h. ( E ) Cell viability (MTT assay 72 h) of A375 clones treated with trametinib (0.001–0.01–0.1–1–10–100 nM). ( F – H ) Western blot analysis of p-ERK, p-AKT, total ERK, and total AKT protein levels in A375 cells treated with trametinib (0.0–0.5–1 nM) for 48 h. All experiments were performed in triplicate. * p ≤ 0.05; ** p ≤ 0.01; ◆◆, §§, ¤¤ = p ≤ 0.01.

Article Snippet: The pLNCX2-PIK3CA wt and pLNCX2-PIK3CA H1047R were obtained by cloning PIK3CA wt and PIK3CA H1047R inserts from pBabe puro HA PIK3CA (Plasmid #12522—Addgene) and pBabe puro HA PIK3CA H1047R (Plasmid #12524—Addgene) plasmids, respectively, into pLNCX2 plasmid.

Techniques: MTT Assay, Western Blot, Clone Assay

Journal: Pharmaceutics

Article Title: The PIK3CA H1047R Mutation Confers Resistance to BRAF and MEK Inhibitors in A375 Melanoma Cells through the Cross-Activation of MAPK and PI3K–Akt Pathways

doi: 10.3390/pharmaceutics14030590

Figure Lengend Snippet: Treatment of A375 pLNCX2 , A375 PIK3CA , and A375 H1047R cells with BYL719. ( A ) Cell viability evaluated by MTT assay after 72 h of treatment with BYL719 (0.001–0.01–0.1–1–10–100 μM). ( B – D ) Western blot analysis of p-ERK, p-Akt, total ERK, and total Akt protein levels in A375 cells treated for 48 h with BYL719 (0.0–0.25–1 μM). All experiments were performed in triplicate. * p ≤ 0.05; ** p ≤ 0.01; ◆ = p ≤ 0.05; ◆◆, ¤¤ = p ≤ 0.01.

Article Snippet: The pLNCX2-PIK3CA wt and pLNCX2-PIK3CA H1047R were obtained by cloning PIK3CA wt and PIK3CA H1047R inserts from pBabe puro HA PIK3CA (Plasmid #12522—Addgene) and pBabe puro HA PIK3CA H1047R (Plasmid #12524—Addgene) plasmids, respectively, into pLNCX2 plasmid.

Techniques: MTT Assay, Western Blot